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United States Patent |
5,547,869
|
Dumas
,   et al.
|
August 20, 1996
|
Plasmids
Abstract
Recombinant plasmids capable of replicating themselves in Escherichia coli
containing a complete or partial sequence of the double or single strand
DNA or a Densovirus causing a densovirosis in a sensitive insect, a
process for obtaining said plasmids and a method of combatting ravager
insects.
Inventors:
|
Dumas; Bruno (Paris, FR);
Gervais; Monica (Saint Leu la Foret, FR);
Bergoin; Max (Saint Christophe lez Ales, FR);
Jourdan; Mireille (Moliere sur Ceze, FR);
Jousset; Francoise X. (Saint Christophe lez Ales, FR)
|
Assignee:
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Roussel Uclaf (FR)
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Appl. No.:
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195814 |
Filed:
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February 14, 1994 |
Foreign Application Priority Data
Current U.S. Class: |
435/252.33; 435/320.1; 536/23.72 |
Intern'l Class: |
C12N 015/00; C12N 015/34; C12N 015/35 |
Field of Search: |
435/320.1,235,252.33,172.3
536/23.72
424/93 A
935/9,32,73,64
|
References Cited
Other References
Bando et al, J. Virol. 61: 553 (1987).
Bolivar et al, Gene 2: 95 (1977).
Beaud et al, Chem. Abstr. 107: 148601v (1987).
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Primary Examiner: Martinell; James
Attorney, Agent or Firm: Bierman and Muserlian
Parent Case Text
PRIOR APPLICATION
This application is a continuation of U.S. patent application Ser. No.
881,054 filed May 11, 1992, now abandoned which is a continuation-in-part
of U.S. patent application Ser. No. 278,735 filed Dec. 2, 1988, now
abandoned.
Claims
What is claimed is:
1. Recombinant plasmids capable of replicating themselves in Escherichia
coli containing a complete sequence of the double or single strand DNA of
a Densovirus (SEQ ID NO. 1) of Junonia (J-DNV) causing a densovirosis in a
sensitive insect.
2. An Escherichia coli host bacterium transformed by a plasmid as defined
in claim 1.
3. A complete sequence of the viral genome of the J-DNV Densovirus
corresponding to sequence SEQ ID No. 1
GTATTGCCA CCGACGACBK CGACGGCAGT CCCTGCCGGC GAAGCCGGCC GCCGCGAAGC 60
GGCAAGGGAC TGCCGTCGTC GTCGTCGGTG GCAATACTCG TAGAGTATAA GCAAGTACTC 120
AGCATGTATA GAGTACTTGA TGACGTCACG GTGACCTTGA CCTTTGACCT TCTATATGAC 180
CTTGACCTAC TTCTGTGACC TTCTGGTCTA CTGACCTTTG ACCTTCTGAG CTGATGTCTA 240
CTGACCTGAT GATGGAGAGG ATCCGAAGAC CTTGGAGCTG AAGGTGGAAC ACAGGACCTG 300
ATGTTAGTGA AGGACAGCAA TAGTGTGCGA GTGAGATAGC ACTATAGCAA CTGTTAGAGC 630
GAGATAGCAA TATGAGTAAA AGAGATAGCA TGCAAACAAA CCTTGAGATA ATTTATGCGC 420
ATTTTATTAT CTTGTTATTG TGACCTCGTT TGACCGGCAA ACTCGCGTCG AGGCTGGGCC 480
GTGTGCAAAA CAGGATGTGG CTGGCCAGCG GACCATTGAC TATATAAAGG CTGACGTGTT 540
CTATTTTTAG TCAGTATGTC TTTCTACACG GCCGGGTTAA TACATCGTGC GCGACCCGGT 600
TATCGTATTA TACCAGAAAG TACTGCTACT GAAGATATTG AACTTGGTGC TATTGGAGAA 660
GAAACTCCAT TATTAAGTGA AGGTGCTGTT ACTGCTGTAG AAGAAAGTGC TGCTGTTGGT 720
TTACCAGAAC TTGGTGCTGG ACTTGCTGGT GCTATTGGAA CACATGCTGA CGTATTGTAT 780
AGAAATAGAA ACGTTTTTAA AAGTGTTTTA ACTGGAAATT ATACCGATTT AAAAGGCAAT 840
CCTTTAAAAC AACGAAACGC TATTTCTGAA AAAACTAAAC AACTTGGAAG AGGAATATTT 900
CAAGGCGATT TCAACCGTGC ATTTCCTGAT GATTTAAAAT TGGAAACTGA ACAAGAAAAA 960
AAAGATTTAC TACGTTATTA TAATCACAAT AGAAGATTAG CTGGATTAAG TGAAGCTTAT 1020
CCACAAGGGA AAGGATACGC TTATGCTAAA AGTCAAAAAG TATTAGAAGC TGAACGACGA 1080
GGATTAACTG TTCCCGGATA TAAATATCTT GGTCCTGGAA ATTCACTTAA CAGAGGTCAA 1140
CCTACTAATC AAATAGACGA AGACGCTAAG GAACACGACG AAGCATACGA TAAAGCGAAA 1200
ACAAGTCAAG AAGTAAGTCA AGCAGATAAT ACATTTGTCA ATAAAGCGTT AGATCACATA 1260
GTTAATGCTA TCAATCTTAA AGAAACTCCT GGTAACGCTT TTGGAGCTGC TATCGGAGCT 1320
ATTGGAATTG GAACTAAGCA AGCTATCGAA AAACACAGTG GAGTAATCTA CCCTTCTGTT 1380
TCAGGTATGT CCCGTCAAAT TAATTCTAAA TACTTAAATA GCTGGCATGA CTGGATTGAG 1440
CAAAATAAAC ATAATAATTT TGAAGGAATA CAATTACCAG AGGACTTTTA CACAGAAGAA 1500
CAAACTCTTT CAGATTCACC GATGTCAGAG GGAACAAAAC GTAAAGCTGA TACTCCTGTT 1560
GAAGAAGGTC CTTCTAAAAA AGGTGCTCAT AACGCTCCAC ATAACTCGCA AGGTACAGAT 1620
CCTCAAAATC CTAGTTCTTC CGGAGCAACT ACTTCTMTTG ACGTTGAAAT GGCTATGTCA 1680
TTACCTGGAA CTGGTTCTGG AACATCATCT GGAGGAGGCA ACACTTCAGG TCAAGAGGTT 1740
TATGTAATTC CTCGTCCATT TTCGAACTTT GGTAAAAAAT TAAGTACTTA TACAAAGTCT 1800
CATAAATTTA TGATATTTGG TCTTGCCAAT AATGTTATTG GACCTACAGG TACTGGTACA 1860
ACAGCTGTAA ATCGTTTAAT TACAACTTGT TTGGCTGAAA TTCCATGGCA GAAATTGCCT 1920
TTGTATATGA ACCAATCTGA ATTTGATTTA TTACCTCCTG GTAGTAGAGT AGTTGAATGT 1980
AATGTTAAAG TAATATTCAG AACTAATCGT ATTGCATTTG AGACTAGTTC AACTGCTACT 2040
AAACAAGCTA CATTGAATCA AATATCTAAT TTACAAACTG CTGTTGGATT AAATAAACTT 2100
GGATGGGGTA TTGATAGATC ATTTACTGCT TTTCAATCAG ATCAACCTAT GATTCCCACT 2160
GCTACTAGTG CACCAAAATA TGAACCTATA ACTGGTACGA CTGGTTATAG AGGTATGATA 2220
GCTGATTATT ATGGTGCTGA TTCTACTAAT GATGCTGCAT TTGGTAATGC TGGTAACTAT 2280
CCTCATCATC AAGTTGGTTC ATTTACTTTT ATTCAAAATT ATTATTGTAT GTATCAACAA 2340
ACCAATCAAG GTACTGGAGG TTGGCCATGT TTAGCTGAAC ATCTTCAACA ATTTGATTCT 2400
AAAACTGTTA ATAATCAATG TTTAATTGAT GTAACTTATA AACCTAAAAT GGGTTTAATT 2460
AAACCACCGT TAAATTATAA AATTATTGGT CAACCTACTG CAAAAGGTAC TATATCTGTT 2520
GGTGATAATT TAGTTAACAT GCGAGGAGCT GTTGTAATAA ATCCACCTGA AGCAACACAA 2580
TCTGTTACTG AATCAACTCA TAATTTGACT CGCAATTTTC CAGCTAATTT GTTTAATATT 2640
TATTCTGACA TTGAAAAATC TCAAATTTTA CATAAAGGAC CTTGGGGACA CGAAAATCCA 2700
CAGATACAAC CAAGTGTTCA TATTGGTATT CAAGCTGTAC CAGCATTAAC TACAGGAGCT 2760
TTACTTGTAA ATTCAAGTCC TTTAAATTCA TGGACTGATT CTATGGGTTA TATTGATGTT 2820
ATGTCTAGTT GTACTGTTAT GGAATCTCAG CCTACACACT TTCCATTTTC GACTGATGCT 2880
AATACTAACC CTGGTAATAC CATTTATCGT ATTAATCTTA CACCGAACTC TCTTACTAGT 2940
GCTTTCAATG GATTGTACGG TAATGGAGCT ACTCTTGGTA ACGTTTAAAT AAAACAATAA 3000
TGTATCCCAT AACCATTTAT TAAAATGTAA TATTATATTT ACTCAATAAA AGGAAAAATG 3060
TCATTGGATG TGGTTTCAAT TCATAATCCT TTAAGAATGG CGCAGCATTC CACTTGTATT 3120
GAATAATTCT ATCACTAAAA GCAGTTTCAT ACATAAAAGG TACAGTATTA TTAGTAAGTA 3180
TTATAACTGG AGTGCGTTTT ACATGTGCAT CCATACGATT TTTAACTCTA ACAGTATAAG 3240
GATCTCCTCC AAACATCATT TTAATTGTAT CAGTTAAAGA ACTCTCATAG TTAGGTTCAT 3300
TCCATAATAA TACACGTTTA TTAGGTGCTT CTTGAAATGC AAACAAGTTA TGTCTATTAG 3360
CTTGACCTAA CTGACCATAA GATAGTAATA ATCCAAAGAT CATATCAAAA AAGAAATTTT 3420
TACCAGCACT TGGAGGAGAT ATAATAAGAA AAGCATTTAA CTTAGGTATA CGACGGTCTA 3480
ATACATTGAC CAAATTAGTA AGAAACTCTA CAATTAAATC TTCATCATCA TTACATTGAA 3540
ATTTAAGTAA TTCTATAATA ATATTCAAAG AATTCTCAAG ATTATCATAT TTCATAGAAG 3600
AAATAAACAA AGCATATGGA TTAAGTTCTT GTTCATCAGT AAAATTATAA TCTTCAGTAA 3660
GTAAATTATA AATTTCTCTC AAAGACATAG CATTTAAATC CTTACCAAAG TCATCGCATG 3720
CTGCTTGTAT ATAATCACGA TTTTTAGGAT CACATAACAA ATCATCATCA CGAAATTCTG 3780
GCACATCACA TATAGCACTC ACTGGAGACA CATAATATTT TCTTAATAAC GCTTTTGTCT 3840
TTTTCCGTAT GTATGCGAAT TTCCCTGCCG AATAGGCTTT CTTTTCATAA AGTCTTCCGT 3900
TAGAACTGCC AGCATCTGAT CTACGGCTAA TTTTGTGCTC TTGCTGTTCA CACTCATAGT 3960
AATCCGTGCA ATCGGAGCTT GATACCATTT CTCTTTCTTT AAACTCTCTG GTCCATCGTA 4020
CACATTCATC GTTACTCGGT ATTTTCCCAC TTTCTCCTCT AACGTATATT GCACGCTCTC 4080
CCCGTTTTCG TACAAAGAAA TAGATGAAGA CATCGTACCA GTCTGTTCGC TTGAATTCCC 4140
AGATGAATTT GACTGGTTTG CCAGTTTTCT GAACAGATCC GAAGGGCTTG ACTTGACTAA 4200
TCCAGATGTC CCTGCAACTG CGATTGGTGT AAGAGCAATC GTGGATGACG TGGATGTGAT 4260
CTCCTTCTTC AGAAAATCCG AACAGTCCGT TTCGTCTACT TCTTCCGTAC TCACGCAAGA 4320
CGTCCAAACA TTGATCACGG AGCTGAATAT CTCGTAAGAT AATGACATCG CTGATATATG 4380
CGCTGGAAGG TTTAATACTT TGCCCAGTAA CGTATCCAAA GAATTCACTG CCCATTTTTT 4440
CCAGTTCTTC TGCCATATAT TGAAAGTTTT CTTGAGATTC TTTTGCAATG CTGGCCATTG 4500
TTCCTCGTCC TCCATGTGAC CATTCACGTT TTCTAGATGT ATTTGCCACC ATGCTACATT 4560
GTTCACTAGG GGATGTTCTT CCTGAAGACT CTCGTATAAT GTCTTGATTA GGTCTGGTAG 4620
TACTATCGGT TTCTCTGTTG GTGTCTCCAT TGTTCATCTG TAATTAATGT CTACTATTAG 4680
GATGTTTTAC ATAAAAACTA TTAGACATAT ATTCGTCCTC TTCTGAACTA CTTGAGTATC 4740
TCCTTTTTTT ATTAGGAGAG TTTTCATAAA TTACAGATAT ATTAAACAAT GGACAAGTAT 4800
GACATATTTG ACACCAACTA CTACCATCTT GTAATCTATT TATAATATCT TCAGCATCTT 4860
CTACGAACAC AGTTCTATTA TACATAAATC TGTATTCATC TGGATCTTCA CAATTAGTAT 4920
AACAAAATTT ACAAATTTTC CACATAGTTT TAAAGGGCTT TCCATTCATA TTCCATGCAT 4980
TAAATTCATC AGTATCATAA GGGTCTCTAT GGTTAATTAA TTCTTTAAAA TAATACACAC 5040
ATTCTATCAG CATAGTTTCA TCTAACCATT CAGGTATTTC ATTTAAATGC ATAACAGCTA 5100
AATATAACTG GAAAGGTAAT CTGTTTTGTT TTGTCCAATC CCAGTGTTCT AATTCTTCCA 5160
TAGCTAAAGT ATGATCTATG TCTTCTCCAC AAGCAATAAT CTGATTATCT AGATCATGCT 5220
GCATATTAAG TATAGGTTTA GGCAAAATTG ACAAGTCTAG ACCATTAAGT CTAGCAGTCT 5280
TATAAGCCTC ATAGAACAAA GACTTTGGAT TGTACACTTT TTCAAATAAA CGAACGAACA 5340
CAAAGAAACC TGGCAATAGA CATACCGATT ATATTCTGGA ACCACTTTTG CACAACACTA 5400
CTTTTTCACT GAGATGTTCA CTCGACGACT GCTGCTCGTA GACTGATGAT GGCGCTCTGC 5460
TGTTATCTCT ATTTATAGCC AATGGTCCGC TGGCCAGCCA CATCCTGTTT TGCACACGGC 5520
CCAGCCTCGA CGCGAGTTTG CCGGTCAAAC GAGGTCACAA TAACAAGATA ATAAAATGCG 5580
CATAAATTAT CTCAAGGTTT GTTTGCATGC TATCTCTTYT ACTCATATTG CTATCTCGCT 5640
CTAACAGTTG CTATAGTGCT ATCTCACTCG CACACTATTG CTGTCCTTCA CTAACATCAG 5700
GTCCTGTGTT CCACCTTCAG CTCCAAGGTC TTCGGATCCT CTCCATCATC AGGTCAGTAG 5760
ACCATCAGCT CAGAAGGTCA AAGGTCAGTA GACCAGAAGG TCACAGAAGT AGGTCAAGGT 5820
CATATAGAAG GTCAAAGGTC AAGGTCACCG TGACGTCATC AAGTACTCTA TACATGCTGA 5880
GTACTTGCTT ATACTCTACG AGTATTGCC (Z).sub.p 5910
wherein N is A or C or G or T bases, wherein N is an integer from 0 to 50
at location 1 and 0 to 130 at location 5910 respectively and M at location
1657 is the base A or C and Y at location 5619 is the base C or T.
4. A sequence of the viral DNA of the Densovirus J-DNV (SEQ ID NO. 1)
having the sequence as defined in claim 3 wherein n is 0 nucleotides in
length.
5. A recombinant plasmid pBRJ capable of replicating itself in E. coli
which contains the complete sequence of the viral genome of the Densovirus
J-DNV as defined in claim 4.
6. An Escherichia coli host bacterium transformed by a plasmid as defined
in claim 5.
Description
STATE OF THE ART
Densonucleoses are diseases affecting a large number of invertebrates,
mainly insects, which are caused by the Densoviruses. The term
densonucleose reflects the hypertrophy characteristic of the nuclei in
which the Densovirus multiplies and under conditions, a mortality
approaching 100% can be reached in 4 or 5 days. These viruses, classified
in the genus Densovirus, constitute, with the two genera Parvovirus and
Dependovirus or parvovirus of vertebrates, the Parvoviridae family.
The Densoviruses are small isometric viruses, not enveloped, with DNA,
strongly pathogenic for many species of ravager insects of economic
importance, notably the lepidoptera, in which they were originally
discovered. In the natural environment, they are responsible for
epizooties which effectively take part in the regulation of the
populations of their hosts. Up to the present, some twenty Densoviruses
have been isolated from insects of different groups: lepidoptera, diptera,
orthoptera and dictyoptera originating from all regions of the globe.
The Densoviruses offer, therefore, very interesting possibilities for use
against insects of economic importance and notably lepidoptera, this new
means of microbiological combat being capable of substituting for chemical
combat or supplementing it. In addition, it is important to emphasize
that, despite their great virulence against insects, preliminary trials
and many years of laboratory experiments have not shown any pathogenic
effect in man. Preparations based on other entomopathogenic viruses such
as the baculovirus are marketed at present.
The methods of industrial production of these viruses, which rely on
infection of insects bred in large quantities, incur high production
costs. Attempts at mass production of these virus in cell cultures have
come up against the same problems of cost. As for the baculoviruses,
multiplication of the Densoviruses is normally obtained on larvae (Ann.
Rev. Entomol., 1979, Vol. 24: pages 63-87), which implies a high
production cost. Furthermore, many strains of Densoviruses cannot be
multiplied on their hosts, taking into account the great difficulties met
in setting up the artificial media necessary for industrial breeding.
It was therefore of great interest to find other ways of production of
infectious material, not requiring the intervention of multiplication on
the host. The possibility exists of obtaining this production of the
Densovirus by genetic engineering and in fact, the size of their genome is
compatible with an insertion in a bacterial plasmid, while this cannot be
envisaged for a virus with a genome of very high molecular weight such as
the baculovirus.
Also relevant prior art is Journal of Virology, Vol. 61 No. 2, February
1987, p. 553-560, Archives of Virology, Vol. 93 No. 1, 2, January, 1987,
p. 139-146 and Comptes rendus de L'Acad. des. Sc. de Paris, Vol. 299 No 20
III (1984), p. 889-894.
OBJECTS OF THE INVENTION
It is an object of the invention to provide novel recombinant plasmids
capable of reproducing themselves in Escherichia coli containing a
complete or partial sequence of double or single strand DNA of a
Densovirus causing a densovirosis in sensitive insect and a method of
producing the said plasmids.
It is another object of the invention to provide a novel method of
combatting ravager insects.
These and other objects and advantages of the invention will become obvious
from the following detailed description.
THE INVENTION
The novel recombinant plasmids of the invention capable of replicating
themselves in Escherichia coli contain a complete or partial sequence of
double or single strand DNA of a Densovirus causing a densovirosis in a
sensitive insect. All the Densoviruses of which a complete or partial
sequence of DNA is contained in the plasmids of the invention are cited in
"Intervirology", 23:61-73 (1985). The Densoviruses of ravager lepidoptera
which have been most studied are the Junonia, Galleria and Agraulis
Densoviruses.
Therefore, the invention is particularly concerned with plasmids that
contain a complete or partial double or single strand sequence of a
Densovirus of Junonia, Galleria or Agraulis and particularly, with the
plasmids that contain a complete or partial sequence of double or single
strand of the DNA of the Densovirus of Junonia (J-DNV). These latter
plasmids are hereafter called pJ in the test.
The genome of the Densovirus is a linear single strand molecule of DNA of
5000 to 6000 bases. During the viral morphogenesis, the virions of the
Densovirus indifferently encapsulate the + chains and the - chains, but
always separately and in equimolecular proportion. Extraction of DNA under
conditions of high ionic strength (0.1 SSC) from such a viral population
leads to the formation of double stranded molecules by the pairing of
complementary chains. [Truffaut et al (1967) and Arch. Ges. Virusforsch.
21, 469-474, and Barvise et al I.O., (1970). FEBS lett. 6, 13-16]. Thus,
by their low molecular weight and their property of forming bicatenary
chains, these genomes lend themselves particularly well to their
integration in plasmids. It must be emphasized that the bicatenary DNA
extracted from the virions is infectious, just as much in cellular
cultures as by transfection of larvae of the sensitive species. [JOUSSET,
1st Congr. Soc. Fr. Microbiol., Toulouse (France), April 1986, CP59, p.
44, and JOUSSET, Soc. Invertebr. Pathol., 1986, p. 121. Colloq.
Invertebr., Veldhoven (The Netherlands), August 1986]. The cloning of the
bicatenary DNA of the Densovirus of Junonia has thus been realized and is
described further on in the experimental part.
The sequence of the cloned genome has been determined by the usual methods
[Sanger et al. (1977) Proc. Nat. Acad. Sci. USA Vol. 74 p. 5463--Maxam et
al (1980), Methods Enzymol Vol. 65 p. 499]. Thus the invention also has as
its subject the complete sequence of the viral genome of the Densovirus
J-DNV corresponding to SEQ ID NO. 1 which is as follows:
NGTATTGCCA CCGACGACGA CGACGGCAGT CCCTGCCGGC GAAGCCGGCC GCCGCGAAGC 60
GGCAAGGGAC TGCCGTCGTC GTCGTCGGTG GCAATACTCG TAGAGTATAA GCAAGTACTC 120
AGCATGTATA GAGTACTTGA TGACGTCACG GTGACCTTGA CCTTTGACCT TCTATATGAC 180
CTTGACCTAC TTCTGTGACC TTCTGGTCTA CTGACCTTTG ACCTTCTGAG CTGATGTCTA 240
CTGACCTGAT GATGGAGAGG ATCCGAAGAC CTTGGAGCTG AAGGTGGAAC ACAGGACCTG 300
ATGTTAGTGA AGGACAGCAA TAGTGTGCGA GTGAGATAGC ACTATAGCAA CTGTTAGAGC 630
GAGATAGCAA TATGAGTAAA AGAGATAGCA TGCAAACAAA CCTTGAGATA ATTTATGCGC 420
ATTTTATTAT CTTGTTATTG TGACCTCGTT TGACCGGCAA ACTCGCGTCG AGGCTGGGCC 480
GTGTGCAAAA CAGGATGTGG CTGGCCAGCG GACCATTGAC TATATAAAGG CTGACGTGTT 540
CTATTTTTAG TCAGTATGTC TTTCTACACG GCCGGGTTAA TACATCGTGC GCGACCCGGT 600
TATCGTATTA TACCAGAAAG TACTGCTACT GAAGATATTG AACTTGGTGC TATTGGAGAA 660
GAAACTCCAT TATTAAGTGA AGGTGCTGTT ACTGCTGTAG AAGAAAGTGC TGCTGTTGGT 720
TTACCAGAAC TTGGTGCTGG ACTTGCTGGT GCTATTGGAA CACATGCTGA CGTATTGTAT 780
AGAAATAGAA ACGTTTTTAA AAGTGTTTTA ACTGGAAATT ATACCGATTT AAAAGGCAAT 840
CCTTTAAAAC AACGAAACGC TATTTCTGAA AAAACTAAAC AACTTGGAAG AGGAATATTT 900
CAAGGCGATT TCAACCGTGC ATTTCCTGAT GATTTAAAAT TGGAAACTGA ACAAGAAAAA 960
AAAGATTTAC TACGTTATTA TAATCACAAT AGAAGATTAG CTGGATTAAG TGAAGCTTAT 1020
CCACAAGGGA AAGGATACGC TTATGCTAAA AGTCAAAAAG TATTAGAAGC TGAACGACGA 1080
GGATTAACTG TTCCCGGATA TAAATATCTT GGTCCTGGAA ATTCACTTAA CAGAGGTCAA 1140
CCTACTAATC AAATAGACGA AGACGCTAAG GAACACGACG AAGCATACGA TAAAGCGAAA 1200
ACAAGTCAAG AAGTAAGTCA AGCAGATAAT ACATTTGTCA ATAAAGCGTT AGATCACATA 1260
GTTAATGCTA TCAATCTTAA AGAAACTCCT GGTAACGCTT TTGGAGCTGC TATCGGAGCT 1320
ATTGGAATTG GAACTAAGCA AGCTATCGAA AAACACAGTG GAGTAATCTA CCCTTCTGTT 1380
TCAGGTATGT CCCGTCAAAT TAATTCTAAA TACTTAAATA GCTGGCATGA CTGGATTGAG 1440
CAAAATAAAC ATAATAATTT TGAAGGAATA CAATTACCAG AGGACTTTTA CACAGAAGAA 1500
CAAACTCTTT CAGATTCACC GATGTCAGAG GGAACAAAAC GTAAAGCTGA TACTCCTGTT 1560
GAAGAAGGTC CTTCTAAAAA AGGTGCTCAT AACGCTCCAC ATAACTCGCA AGGTACAGAT 1620
CCTCAAAATC CTAGTTCTTC CGGAGCAACT ACTTCTMTTG ACGTTGAAAT GGCTATGTCA 1680
TTACCTGGAA CTGGTTCTGG AACATCATCT GGAGGAGGCA ACACTTCAGG TCAAGAGGTT 1740
TATGTAATTC CTCGTCCATT TTCGAACTTT GGTAAAAAAT TAAGTACTTA TACAAAGTCT 1800
CATAAATTTA TGATATTTGG TCTTGCCAAT AATGTTATTG GACCTACAGG TACTGGTACA 1860
ACAGCTGTAA ATCGTTTAAT TACAACTTGT TTGGCTGAAA TTCCATGGCA GAAATTGCCT 1920
TTGTATATGA ACCAATCTGA ATTTGATTTA TTACCTCCTG GTAGTAGAGT AGTTGAATGT 1980
AATGTTAAAG TAATATTCAG AACTAATCGT ATTGCATTTG AGACTAGTTC AACTGCTACT 2040
AAACAAGCTA CATTGAATCA AATATCTAAT TTACAAACTG CTGTTGGATT AAATAAACTT 2100
GGATGGGGTA TTGATAGATC ATTTACTGCT TTTCAATCAG ATCAACCTAT GATTCCCACT 2160
GCTACTAGTG CACCAAAATA TGAACCTATA ACTGGTACGA CTGGTTATAG AGGTATGATA 2220
GCTGATTATT ATGGTGCTGA TTCTACTAAT GATGCTGCAT TTGGTAATGC TGGTAACTAT 2280
CCTCATCATC AAGTTGGTTC ATTTACTTTT ATTCAAAATT ATTATTGTAT GTATCAACAA 2340
ACCAATCAAG GTACTGGAGG TTGGCCATGT TTAGCTGAAC ATCTTCAACA ATTTGATTCT 2400
AAAACTGTTA ATAATCAATG TTTAATTGAT GTAACTTATA AACCTAAAAT GGGTTTAATT 2460
AAACCACCGT TAAATTATAA AATTATTGGT CAACCTACTG CAAAAGGTAC TATATCTGTT 2520
GGTGATAATT TAGTTAACAT GCGAGGAGCT GTTGTAATAA ATCCACCTGA AGCAACACAA 2580
TCTGTTACTG AATCAACTCA TAATTTGACT CGCAATTTTC CAGCTAATTT GTTTAATATT 2640
TATTCTGACA TTGAAAAATC TCAAATTTTA CATAAAGGAC CTTGGGGACA CGAAAATCCA 2700
CAGATACAAC CAAGTGTTCA TATTGGTATT CAAGCTGTAC CAGCATTAAC TACAGGAGCT 2760
TTACTTGTAA ATTCAAGTCC TTTAAATTCA TGGACTGATT CTATGGGTTA TATTGATGTT 2820
ATGTCTAGTT GTACTGTTAT GGAATCTCAG CCTACACACT TTCCATTTTC GACTGATGCT 2880
AATACTAACC CTGGTAATAC CATTTATCGT ATTAATCTTA CACCGAACTC TCTTACTAGT 2940
GCTTTCAATG GATTGTACGG TAATGGAGCT ACTCTTGGTA ACGTTTAAAT AAAACAATAA 3000
TGTATCCCAT AACCATTTAT TAAAATGTAA TATTATATTT ACTCAATAAA AGGAAAAATG 3060
TCATTGGATG TGGTTTCAAT TCATAATCCT TTAAGAATGG CGCAGCATTC CACTTGTATT 3120
GAATAATTCT ATCACTAAAA GCAGTTTCAT ACATAAAAGG TACAGTATTA TTAGTAAGTA 3180
TTATAACTGG AGTGCGTTTT ACATGTGCAT CCATACGATT TTTAACTCTA ACAGTATAAG 3240
GATCTCCTCC AAACATCATT TTAATTGTAT CAGTTAAAGA ACTCTCATAG TTAGGTTCAT 3300
TCCATAATAA TACACGTTTA TTAGGTGCTT CTTGAAATGC AAACAAGTTA TGTCTATTAG 3360
CTTGACCTAA CTGACCATAA GATAGTAATA ATCCAAAGAT CATATCAAAA AAGAAATTTT 3420
TACCAGCACT TGGAGGAGAT ATAATAAGAA AAGCATTTAA CTTAGGTATA CGACGGTCTA 3480
ATACATTGAC CAAATTAGTA AGAAACTCTA CAATTAAATC TTCATCATCA TTACATTGAA 3540
ATTTAAGTAA TTCTATAATA ATATTCAAAG AATTCTCAAG ATTATCATAT TTCATAGAAG 3600
AAATAAACAA AGCATATGGA TTAAGTTCTT GTTCATCAGT AAAATTATAA TCTTCAGTAA 3660
GTAAATTATA AATTTCTCTC AAAGACATAG CATTTAAATC CTTACCAAAG TCATCGCATG 3720
CTGCTTGTAT ATAATCACGA TTTTTAGGAT CACATAACAA ATCATCATCA CGAAATTCTG 3780
GCACATCACA TATAGCACTC ACTGGAGACA CATAATATTT TCTTAATAAC GCTTTTGTCT 3840
TTTTCCGTAT GTATGCGAAT TTCCCTGCCG AATAGGCTTT CTTTTCATAA AGTCTTCCGT 3900
TAGAACTGCC AGCATCTGAT CTACGGCTAA TTTTGTGCTC TTGCTGTTCA CACTCATAGT 3960
AATCCGTGCA ATCGGAGCTT GATACCATTT CTCTTTCTTT AAACTCTCTG GTCCATCGTA 4020
CACATTCATC GTTACTCGGT ATTTTCCCAC TTTCTCCTCT AACGTATATT GCACGCTCTC 4080
CCCGTTTTCG TACAAAGAAA TAGATGAAGA CATCGTACCA GTCTGTTCGC TTGAATTCCC 4140
AGATGAATTT GACTGGTTTG CCAGTTTTCT GAACAGATCC GAAGGGCTTG ACTTGACTAA 4200
TCCAGATGTC CCTGCAACTG CGATTGGTGT AAGAGCAATC GTGGATGACG TGGATGTGAT 4260
CTCCTTCTTC AGAAAATCCG AACAGTCCGT TTCGTCTACT TCTTCCGTAC TCACGCAAGA 4320
CGTCCAAACA TTGATCACGG AGCTGAATAT CTCGTAAGAT AATGACATCG CTGATATATG 4380
CGCTGGAAGG TTTAATACTT TGCCCAGTAA CGTATCCAAA GAATTCACTG CCCATTTTTT 4440
CCAGTTCTTC TGCCATATAT TGAAAGTTTT CTTGAGATTC TTTTGCAATG CTGGCCATTG 4500
TTCCTCGTCC TCCATGTGAC CATTCACGTT TTCTAGATGT ATTTGCCACC ATGCTACATT 4560
GTTCACTAGG GGATGTTCTT CCTGAAGACT CTCGTATAAT GTCTTGATTA GGTCTGGTAG 4620
TACTATCGGT TTCTCTGTTG GTGTCTCCAT TGTTCATCTG TAATTAATGT CTACTATTAG 4680
GATGTTTTAC ATAAAAACTA TTAGACATAT ATTCGTCCTC TTCTGAACTA CTTGAGTATC 4740
TCCTTTTTTT ATTAGGAGAG TTTTCATAAA TTACAGATAT ATTAAACAAT GGACAAGTAT 4800
GACATATTTG ACACCAACTA CTACCATCTT GTAATCTATT TATAATATCT TCAGCATCTT 4860
CTACGAACAC AGTTCTATTA TACATAAATC TGTATTCATC TGGATCTTCA CAATTAGTAT 4920
AACAAAATTT ACAAATTTTC CACATAGTTT TAAAGGGCTT TCCATTCATA TTCCATGCAT 4980
TAAATTCATC AGTATCATAA GGGTCTCTAT GGTTAATTAA TTCTTTAAAA TAATACACAC 5040
ATTCTATCAG CATAGTTTCA TCTAACCATT CAGGTATTTC ATTTAAATGC ATAACAGCTA 5100
AATATAACTG GAAAGGTAAT CTGTTTTGTT TTGTCCAATC CCAGTGTTCT AATTCTTCCA 5160
TAGCTAAAGT ATGATCTATG TCTTCTCCAC AAGCAATAAT CTGATTATCT AGATCATGCT 5220
GCATATTAAG TATAGGTTTA GGCAAAATTG ACAAGTCTAG ACCATTAAGT CTAGCAGTCT 5280
TATAAGCCTC ATAGAACAAA GACTTTGGAT TGTACACTTT TTCAAATAAA CGAACGAACA 5340
CAAAGAAACC TGGCAATAGA CATACCGATT ATATTCTGGA ACCACTTTTG CACAACACTA 5400
CTTTTTCACT GAGATGTTCA CTCGACGACT GCTGCTCGTA GACTGATGAT GGCGCTCTGC 5460
TGTTATCTCT ATTTATAGCC AATGGTCCGC TGGCCAGCCA CATCCTGTTT TGCACACGGC 5520
CCAGCCTCGA CGCGAGTTTG CCGGTCAAAC GAGGTCACAA TAACAAGATA ATAAAATGCG 5580
CATAAATTAT CTCAAGGTTT GTTTGCATGC TATCTCTTYT ACTCATATTG CTATCTCGCT 5640
CTAACAGTTG CTATAGTGCT ATCTCACTCG CACACTATTG CTGTCCTTCA CTAACATCAG 5700
GTCCTGTGTT CCACCTTCAG CTCCAAGGTC TTCGGATCCT CTCCATCATC AGGTCAGTAG 5760
ACCATCAGCT CAGAAGGTCA AAGGTCAGTA GACCAGAAGG TCACAGAAGT AGGTCAAGGT 5820
CATATAGAAG GTCAAAGGTC AAGGTCACCG TGACGTCATC AAGTACTCTA TACATGCTGA 5880
GTACTTGCTT ATACTCTACG AGTATTGCCN (SEQ ID NO. 1) 5910
This represents the complete nucleotide sequence of the viral genome of the
Densovirus J-DNV. N is the bases A or C or G or T wherein N is 0 to 50
nucleotides in length at location 1 and N is 0 to 130 nucleotides in
length at location 5910. M is A or C and Y is C or T.
Also a part of the invention concepts the complete sequence of viral DNA of
the Densovirus J-DNV corresponding to the sequence given above (SEQ ID NO.
1) wherein the two extremities N are eliminated.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-1C show the viral genome of the Densovirus J-DNV.
FIG. 2 shows a restriction map of Densovirus of Junonia.
FIG. 3 shows the construction of pBRJ.
The invention is concerned particularly with the plasmids characterized in
that they contain the complete sequence of the viral genome of the
Densovirus J-DNV given above as well as the plasmids characterized in that
they contain the complete or partial sequence of the double or single
strand DNA of the Densovirus of Galleria. These plasmids are called pJ
hereafter in the text. Among the pJ vectors defined above, the vector pBRJ
is the preferred one. This plasmid contains the complete sequence of the
viral DNA of the cloned Densovirus J-DNV corresponding to the sequence
given above in which the two extremities N are eliminated. The principle
of construction of the plasmid is given further on in the text and its
construction is detailed in the experimental part.
The construction of the plasmids of the invention uses the known techniques
utilized in this field. These techniques are described, for example, in
Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory (1982) or in "Basic methods in molecular biology",
Davis--Dibner Battney--Elsevier (1986).
The starting vector used in the construction of the plasmids of the
invention is preferably pBR 322, but any other classical cloning vector
can be used, such as for example, the vector PUC.sub.18 [Norrander, J. et
al., Gene 26, 101-106 (1983)] or the vector pAT.sub.153 [Twigg, A. J. and
Sheratt D., Nature 283, 216-218 (1980)[.
The starting vector is digested by one or more restriction enzymes in a
cloning site which is preferably a unique site. The choice of this site is
dictated by prior knowledge concerning the cartography of the viral genome
of the Densovirus. Thus, the pJ plasmids previously defined can be
constructed from the starting vector pBR 322 digested by the enzymes
generating free ends as, for example, ECoR V or NrUI or the enzymes
generating sticky ends as, for example, PstI. These various possibilities
derive from the cartography of the viral genome J-DNV given in FIG. 2. The
same cloning strategy can, naturally, be applied to the other plasmids of
the invention such as, for example, to the previously defined pJ plasmids
which are constructed using the restriction enzyme PstI.
The construction of the preferred plasmid of the invention, the pBRJ
plasmid, is detailed in FIG. 3. The viral DNA has been obtained from the
Junonia virus by known techniques of extraction and purification. The
purified DNA, after treatment with polymerase, is integrated at the unique
site ECoRV of linearized pBR322. After transformation in Escherichia coil,
only those colonies resistant to ampicillin and sensitive to tetracycline
are selected.
Mini-lyses of recombinant bacteria are carried out to retain only the
colonies containing the plasmids having inserted a fragment of size near
to that of the genomic DNA. The scheme of construction of pBRJ is given in
FIG. 3.
The invention is further concerned with the Escherichia coli host bacteria
transformed by the recombinant plasmids capable of replicating themselves
in Escherichia coli, characterized in that the contain a complete or
partial sequence of double or single strand DNA of a Densovirus causing a
densovirosis in a sensitive insect. It concerns particularly the bacteria
transformed by the pJ plasmids and quite particularly by the pBRJ plasmid.
All the plasmids as defined above, and notably the pJ plasmids, and among
these the plasmid pBRJ, can be used to combat ravager insects. The tests
described in the experimental part show that the transfection by injection
of the pBRJ plasmid in the hemolymph of larvae of Spodoptera enables the
reproduction of a densovirosis identical to that caused by inoculation of
the virion or of the viral J DNA. The same sensitivity to pBRJ has been
shown on strains of Spodoptera resistant to chemical insecticides such as
deltamethrine.
The invention is thus concerned with the method of combatting ravager
insects biologically with the plasmids of the invention, and particularly
the pJ plasmids, and among these, the plasmid pBRJ.
The insects of economic importance concerned in such usage are certain
dipter vectors of medical and veterinary interest, and lepidopters and
coleopters which are ravagers of food-producing and industrial crops such
as cotton, corn, soya, oil-palm and bananas.
The invention also includes all the insecticidal formulations adapted for
such use. The suitable formulations are those which enable the DNA to be
coated with the plasmids of the invention. The vesicules coating this DNA
must respond to several criteria: 1) hydrophilic internal cavity of
sufficient diameter, 2) ensuring the protection of the DNA of the plasmid
in the intestinal tract and 3) liberating their contents at the target
cells, for example, those of the mid-intestine. Such vesicles are, for
example, niosomes, liposomes, microcapsules of gelatin or of hydrosoluble
polymers or of acrylamide or of any other type of microcapsule compatible
with the criteria defined above. Forms of encapsulation in monocatenary
phages of the M 13 type can also be envisaged.
The method of the invention for combatting ravager insect biologically
comprises administering by ingestion an insecticidally effective amount of
a plasmid of the invention.
In the following examples there are described several preferred embodiments
to illustrate the invention but it should be understood that the invention
is not intended to be limited to the specific embodiments.
EXAMPLE
Construction of the pBRJ plasmid
1) Purification of the DNA of the virus.
2) Cloning the DNA of the virus.
3) Sequence of the DNA of the virus.
4) Results of the transfections of the larvae of Spodoptera littoralis by
the DNA of the Densovirus cloned or not in pBR 322.
Purification of the DNA
a) Purification of the Virus
20 to 30 Spodoptera littoralis larvae infected by the Densovirus of Junonia
were ground up in a Potter dish in Tris HCl 0.1M pH 7.4 ascorbic acid 2%
buffer and the product was clarified by centrifuging for 10 minutes at
10,000 g. The virions ere then agglomerated by centrifuging for 90 minutes
at 35,000 rpm in an SW 41 rotor at 4.degree. C. and the residue was taken
up and deposited on a Renografin 20-76% gradient and centrifuged for 15
hours at 35,000 rpm in an SW 41 rotor. The band of virus localized in the
lower third of the gradient was recovered and then dialyzed against buffer
TE (tris HCl 10 mM EDTA 1 mM pH 8.0) for 3 days with a buffer change every
12 hours. The purity of the viral suspensions was then checked by
electronic microscopy and UV spectrophotometry and their concentration was
estimated by measurement of the optical density at 260 nm. One unit of
optical density at 260 nm is equivalent to 100 .mu.g of virion per ml.
b) Extraction of the DNA
The buffer of the viral suspension was adjusted to 4 mM EDTA at 100 .mu.g
of K proteinase per ml and at 2% sarkosyl. After an incubation of 2 hours
at 37.degree. C., the suspension was extracted three times with the same
volume of phenol saturated with TE buffer. The aqueous phase was recovered
and dialyzed against TE buffer. The DNA was then precipitated by addition
of sodium acetate or lithium chloride at 0.2M final and two volumes of
ethanol from 12 hours to 16 hours at -20.degree. C. The DNA residue was
rinsed three times with 70% alcohol and then taken up in TE buffer. The
purity of the DNA viral solution was checked and its concentration
determined by UV spectrometry.
2) Cloning the DNA of the Virus
a) Preparation of the DNA of the Virus
The extremities of the virus were repaired by the DNA polymerase of E. coli
Klenow fragment. 3 .mu.g of DNA of the virus were repaired in a volume of
75 .mu.l with 7.5 U of DNA polymerase of E. coli (Boehringer Mannheim
Biochemicals--BMB cat. No. 1014531). The repair buffer contained
deoxyadenosine, deoxycytosine, guanosine and thymidine at a concentration
of 40 uM, some MgCl.sub.2 5 mM, betamercaptoethanol 10 mM, beef albumin
serum 100 ug/ml and Tris HCl 10 mM pH 7.5. The reaction mixture was
incubated at 25.degree. C. for one hour and after one hour at 65.degree.
C., the mixture was extracted with phenol-saturated Tris pH 78.5 0.1M,
then precipitated in the presence of one volume of 0.3M acetate and two
volumes of 100% ethanol.
b) Preparation of the DNA of the pBR322 Vector
The DNA of pBR322 was prepared by the method described by Maniatis et al
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory
(1982). 2.5 .mu.g of DNA of pBR322 were digested by the restriction enzyme
EcoRV under the conditions described by the BMB supplier using 18 U/.mu.g
of DNA of enzyme in a volume of 50 .mu.l over 4 hours at 37.degree. C. The
digestion of the DNA was verified on a 1% agarose gel and the DNA was then
dephosphorylated with alkaline phosphatase from the intestines of a calf.
2 .mu.l of alkaline phosphatase BMB, cat. No. 713023 at 22 U/.mu.l were
added and after incubating for one hour at 37.degree. C., the reaction was
stopped by incubating for 45 minutes at 65.degree. C. After extracting
with saturated phenol Tris 0.1M pH 7.5, the aqueous phase was precipitated
with ethanol in the presence of 0.3M acetate.
c) Ligation of the DNA of the Junonia Virus on the pBR322 Vector Linearized
by the Restriction Enzyme EcoRV
After precipitation and drying, the DNA of the vector was taken up at a
concentration of 40 ng/.mu.l and the DNA of the virus was taken up at a
concentration of 1 .mu.g/.mu.l. The ligation was effected in a volume of
25 .mu.l under the conditions described by the manufacturer (New England
Biolabs). After incubating for one night at 4.degree. C., the mixture was
ready to transform the Escherichia coli bacteria (MC1061). (Casabadan M.
J. Methods in Enzymol, (1983) 100 p. 293-3080).
d) Transformation of the Competent Bacteria
The competent bacteria were prepared by the procedure of Hanahan (Maniatis
1982) and preserved at -70.degree. C. in the form of fractions of 200
.mu.l. To 100 .mu.l of competent bacteria thawed at 37.degree. C., without
exceeding 0.degree. C., 1 .mu.l of the preceding solution containing the
DNA of the virus and the vector pBR322 were added.
After incubating for 15 minutes at 0.degree. C. followed by a thermal shock
of 5.degree. to 37.degree. C., there were added 900 ul of L B medium (10 g
Bactotryptone DIFCO, 5 g of yeast extract DIFCO, 5 g of NaCl per liter,
autoclaved at 120.degree. C.). Recombinant bacteria were selected on
dishes of L B agar medium containing 100 .mu.g/ml of ampicillin. Out of
253 colonies resistant to ampicillin, 50 colonies sensitive to
tetracycline were chosen.
e) Mini-lysis of the Recombinant Bacteria and Obtaining the pBRJ
Among these recombinant bacteria sensitive to tetracycline, only one
carried the DNA of the cloned virus at the EcoRV site. Starting from a
culture (1.5 ml) in L-B medium of each of the recombinant clones, the
plasmidic DNA's were prepared by the alkaline method (Maniatis 1982). Each
of the DNA's had been digested by the restriction enzyme EcoR1. One colony
only carries a plasmidic DNA corresponding to a DNA of pBR322 with Junonia
Densovirus inserted at the EcoRV site destroyed during the cloning. The
corresponding pBRJ plasmid was prepared in a large quantity from a liter
of culture at saturation in L-B medium by the method with sodium hydroxide
and sodium dodecyl sulfate (Maniatis 1982). Digestions by the restriction
enzymes BamH1, Pvu2, Bg12, Nru1, Acc1, Pvu1, BstE2, Sph1, EcoR1, Hind3,
Rsa1, Bg11 enabled verification that the DNA of the Densovirus was
inserted at the EcoRV site destroyed during the cloning in the orientation
described in FIG. 3.
3) Sequence of the DNA of the Junonia Densovirus
The sequence of the DNA of the virus was determined for its greater part by
Sanger's method (Sanger 1977). The junction virus pBR322 on the side of
the Hind 3 site of PBR 322 was sequenced by the method of Maxam and
Gilbert.
a) Sanger's Method
1) Random Cloning
Enzymatic Method
Random clonings were realized with the restriction enzymes:
Sau3a1, Xho2, Alu1, Hae3, Acc1. The DNA of the virus was digested by these
restriction enzymes which cut the DNA of the Junonia Densovirus frequently
to obtain fragments of the order of 100 pairs of bases (pb) at 1000 pb.
The fragments obtained were sub-cloned in the vectors M13mp8 and
M13mp9(Messing, J. and Viera, Gene, 19, 259-268 (1982) digested by the
enzyme BamH1 (digestions Sau3A, Xho2), by the enzyme Sma1 (digestions
Alu1, Hae3) and by the enzyme Acc1 (digestion Acc1).
Sonication Method
The DNA of the virus was sonicated until fragments on the order of 500 pb
were obtained and the fragments obtained were repaired by the DNA
polymerase of E. coli Klenow fragment by the process previously described.
The fragments obtained were then sub-cloned in the vectors M13mp8 and
M13mp9 digested by the enzyme Sma1.
2) Sub-cloning of Fragments Identified from the Restriction Cartography
Sub-cloning was done of the identified restriction fragments of which the
size was less than 2000 pb obtained by digestions with the restriction
enzymes (see restriction cartography in FIG. 1. BamH1, EcoR1, Hind3-BstE2,
BstE2-BamH1, BstE2-Hind3, Hind3-Pvu2, Xba1-BstE2, Xba1, Hae3, Pvu2-Hae3,
EcoR1-Xba1, Xba1-BstE2, Hae3-EcoR1, Hae3-Xba1, EcoR1-Pvu2, He3-BstE2. The
fragments with a molecular weight of 110 pb to 2000 pb obtained by
digestion with the above restriction enzymes were sub-cloned and then
sequenced step by step using primers [Sanger (1977) Proc. Nat. Acad. Sci.
USA Vol. 74 p. 5463; Biggin (1983) Proc. Nat. Acad. Sci. Vol. 80 p. 3963].
b) Method of Maxam and Gilbert
A fragment of 375 pb Hind 3 and BsteII near to the EcoR1 site of pBR322
proved to be impossible to sequence using Sanger's method. The sequence of
this fragment was obtained from the Hind3 site and the BstE2 site by
marking with T4 DNA polynucleotide kinase and ATP 32 P or with the DNA
polymerase Klenow fragment and dTT.sup.32 p or dCT.sup.32 p (Maxam and
Gilbert). The reactions were set to migrate on an acrylamide denaturising
gel, urea [Sanger et al (1978), F.E.B.S. lett. Vol. 87, p. 107) in the
presence of formamide at 40%.
c) Organization of the Sequences
The sequences obtained were entered in a computer of type Microvax2 and
they were compared with the help of programs of the DB SYSTEM written by
R. STADEN. [Nucleic Acids Research 12 (1) June 1987, p. 387-395]. After
editing the sequences with the help of the edition program EDIT under
VAX/VMS, the sequences were compared with the help of STADEN'S DBCOMP
program and then they were organized with the help of the program DBUTIL.
The sequence consensus obtained was that set forth above (SEQ ID NO. 1).
4) Results of the Transfections of the Larvae of Spodoptera Littoralis by
DNA of the Densovirus Cloned or Not in pBR322
a) Process of Transfection by Inoculation
Different concentrations of DNA J or of pBRJ were inoculated to the young
larvae of Spodoptera littoralis (stage 2 or 3 ) in a TE buffer, pH 8.0
(Tris 10 mM, EDTA 1 mM) with DEAE-dextran added (2 mg/ml final).
b) Surveillance of the Larvae
The transfected larvae and those of the control lots were raised
individually on axenic medium at 25.degree. C. Each day the dead larvae
were removed, the data of nymphosis was noted and the nymphs were removed
6 days after nymphosis.
c) Control of the Transfected Insects
Each individual was ground up individually in 1 ml of PBS +2% of sodium
azide and the grindings were clarified and the presence of virus was
determined in the supernatants by the indirect ELISA method using
anti-bodies titrated against Junonia Densovirus prepared on mice and
anti-IgG of mice marked with peroxidase. Certain samples were checked with
the electronic microscope.
d) Results
The transfection of the young larvae of Spodoptera littoralis by
inoculation of DNAJ or DNA of pBRJ at concentrations of 30 ng of ADN-J per
larva and of 56 ng of pBRJ/larva led to an infection percentage greater
than 80% in the two cases. Comparable results were obtained by
transfecting larvae of Spodoptera littoralis of Madagascan origin
resistant to delta-methrin. Moreover, no infection appeared in the control
lots inoculated either with the extraction buffer TE or with DNA pBR322 at
a concentration of 30 ng to 1 ng/larva.
Example of Insecticide Composition
A composition of Liposome with base of 5 mg of phosphatidyl choline, 5 mg
of phosphatidyl serine, 2.5 mg of cholesterol and 28 .mu.g of pBRJ and
sufficient Water q.s.p. of 1 ml was prepared.
Various modifications of the plasmids and methods of the invention may be
made without departing from the spirit or scope thereof and it is to be
understood that the invention is intended to be limited only as defined in
the appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 1
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5910
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: UNKNOWN
(D) TOPOLOGY: UNKNOWN
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: DENSOVIRUS
(B) STRAIN: DENSOVIRUS OF JUNONIA
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE: LARVAE
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: SPODOPTERA LITTORALIS
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(B) LOCATION: 1
(D) OTHER INFORMATION: N IS A OR C OR G OR T,
WHEREIN N IS ZERO TO 50 NUCLEOTIDES IN LENGTH
(ix) FEATURE:
(B) LOCATION: 1657
(D) OTHER INFORMATION: M IS A OR C
(ix) FEATURE:
(B) LOCATION: 5619
(D) OTHER INFORMATION: Y IS C OR T
(ix) FEATURE:
(B) LOCATION: 5910
(D) OTHER INFORMATION: N IS A OR C OR G OR T,
WHEREIN N IS ZERO TO 130 NUCLEOTIDES IN LENGTH
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
NGTATTGCCACCGACGACGACGACGGCAGTCCCTGCCGGCGAAGCCGGCCGCCGCGAAGC60
GGCAAGGGACTGCCGTCGTCGTCGTCGGTGGCAATACTCGTAGAGTATAAGCAAGTACTC120
AGCATGTATAGAGTACTTGATGACGTCACGGTGACCTTGACCTTTGACCTTCTATATGAC180
CTTGACCTACTTCTGTGACCTTCTGGTCTACTGACCTTTGACCTTCTGAGCTGATGTCTA240
CTGACCTGATGATGGAGAGGATCCGAAGACCTTGGAGCTGAAGGTGGAACACAGGACCTG300
ATGTTAGTGAAGGACAGCAATAGTGTGCGAGTGAGATAGCACTATAGCAACTGTTAGAGC360
GAGATAGCAATATGAGTAAAAGAGATAGCATGCAAACAAACCTTGAGATAATTTATGCGC420
ATTTTATTATCTTGTTATTGTGACCTCGTTTGACCGGCAAACTCGCGTCGAGGCTGGGCC480
GTGTGCAAAACAGGATGTGGCTGGCCAGCGGACCATTGACTATATAAAGGCTGACGTGTT540
CTATTTTTAGTCAGTATGTCTTTCTACACGGCCGGGTTAATACATCGTGCGCGACCCGGT600
TATCGTATTATACCAGAAAGTACTGCTACTGAAGATATTGAACTTGGTGCTATTGGAGAA660
GAAACTCCATTATTAAGTGAAGGTGCTGTTACTGCTGTAGAAGAAAGTGCTGCTGTTGGT720
TTACCAGAACTTGGTGCTGGACTTGCTGGTGCTATTGGAACACATGCTGACGTATTGTAT780
AGAAATAGAAACGTTTTTAAAAGTGTTTTAACTGGAAATTATACCGATTTAAAAGGCAAT840
CCTTTAAAACAACGAAACGCTATTTCTGAAAAAACTAAACAACTTGGAAGAGGAATATTT900
CAAGGCGATTTCAACCGTGCATTTCCTGATGATTTAAAATTGGAAACTGAACAAGAAAAA960
AAAGATTTACTACGTTATTATAATCACAATAGAAGATTAGCTGGATTAAGTGAAGCTTA1020
CCACAAGGGAAAGGATACGCTTATGCTAAAAGTCAAAAAGTATTAGAAGCTGAACGACG1080
GGATTAACTGTTCCCGGATATAAATATCTTGGTCCTGGAAATTCACTTAACAGAGGTCA1140
CCTACTAATCAAATAGACGAAGACGCTAAGGAACACGACGAAGCATACGATAAAGCGAA1200
ACAAGTCAAGAAGTAAGTCAAGCAGATAATACATTTGTCAATAAAGCGTTAGATCACAT1260
GTTAATGCTATCAATCTTAAAGAAACTCCTGGTAACGCTTTTGGAGCTGCTATCGGAGC1320
ATTGGAATTGGAACTAAGCAAGCTATCGAAAAACACAGTGGAGTAATCTACCCTTCTGT1380
TCAGGTATGTCCCGTCAAATTAATTCTAAATACTTAAATAGCTGGCATGACTGGATTGA1440
CAAAATAAACATAATAATTTTGAAGGAATACAATTACCAGAGGACTTTTACACAGAAGA1500
CAAACTCTTTCAGATTCACCGATGTCAGAGGGAACAAAACGTAAAGCTGATACTCCTGT1560
GAAGAAGGTCCTTCTAAAAAAGGTGCTCATAACGCTCCACATAACTCGCAAGGTACAGA1620
CCTCAAAATCCTAGTTCTTCCGGAGCAACTACTTCTMTTGACGTTGAAATGGCTATGTC1680
TTACCTGGAACTGGTTCTGGAACATCATCTGGAGGAGGCAACACTTCAGGTCAAGAGGT1740
TATGTAATTCCTCGTCCATTTTCGAACTTTGGTAAAAAATTAAGTACTTATACAAAGTC1800
CATAAATTTATGATATTTGGTCTTGCCAATAATGTTATTGGACCTACAGGTACTGGTAC1860
ACAGCTGTAAATCGTTTAATTACAACTTGTTTGGCTGAAATTCCATGGCAGAAATTGCC1920
TTGTATATGAACCAATCTGAATTTGATTTATTACCTCCTGGTAGTAGAGTAGTTGAATG1980
AATGTTAAAGTAATATTCAGAACTAATCGTATTGCATTTGAGACTAGTTCAACTGCTAC2040
AAACAAGCTACATTGAATCAAATATCTAATTTACAAACTGCTGTTGGATTAAATAAACT2100
GGATGGGGTATTGATAGATCATTTACTGCTTTTCAATCAGATCAACCTATGATTCCCAC2160
GCTACTAGTGCACCAAAATATGAACCTATAACTGGTACGACTGGTTATAGAGGTATGAT2220
GCTGATTATTATGGTGCTGATTCTACTAATGATGCTGCATTTGGTAATGCTGGTAACTA2280
CCTCATCATCAAGTTGGTTCATTTACTTTTATTCAAAATTATTATTGTATGTATCAACA2340
ACCAATCAAGGTACTGGAGGTTGGCCATGTTTAGCTGAACATCTTCAACAATTTGATTC2400
AAAACTGTTAATAATCAATGTTTAATTGATGTAACTTATAAACCTAAAATGGGTTTAAT2460
AAACCACCGTTAAATTATAAAATTATTGGTCAACCTACTGCAAAAGGTACTATATCTGT2520
GGTGATAATTTAGTTAACATGCGAGGAGCTGTTGTAATAAATCCACCTGAAGCAACACA2580
TCTGTTACTGAATCAACTCATAATTTGACTCGCAATTTTCCAGCTAATTTGTTTAATAT2640
TATTCTGACATTGAAAAATCTCAAATTTTACATAAAGGACCTTGGGGACACGAAAATCC2700
CAGATACAACCAAGTGTTCATATTGGTATTCAAGCTGTACCAGCATTAACTACAGGAGC2760
TTACTTGTAAATTCAAGTCCTTTAAATTCATGGACTGATTCTATGGGTTATATTGATGT2820
ATGTCTAGTTGTACTGTTATGGAATCTCAGCCTACACACTTTCCATTTTCGACTGATGC2880
AATACTAACCCTGGTAATACCATTTATCGTATTAATCTTACACCGAACTCTCTTACTAG2940
GCTTTCAATGGATTGTACGGTAATGGAGCTACTCTTGGTAACGTTTAAATAAAACAATA3000
TGTATCCCATAACCATTTATTAAAATGTAATATTATATTTACTCAATAAAAGGAAAAAT3060
TCATTGGATGTGGTTTCAATTCATAATCCTTTAAGAATGGCGCAGCATTCCACTTGTAT3120
GAATAATTCTATCACTAAAAGCAGTTTCATACATAAAAGGTACAGTATTATTAGTAAGT3180
TTATAACTGGAGTGCGTTTTACATGTGCATCCATACGATTTTTAACTCTAACAGTATAA3240
GATCTCCTCCAAACATCATTTTAATTGTATCAGTTAAAGAACTCTCATAGTTAGGTTCA3300
TCCATAATAATACACGTTTATTAGGTGCTTCTTGAAATGCAAACAAGTTATGTCTATTA3360
CTTGACCTAACTGACCATAAGATAGTAATAATCCAAAGATCATATCAAAAAAGAAATTT3420
TACCAGCACTTGGAGGAGATATAATAAGAAAAGCATTTAACTTAGGTATACGACGGTCT3480
ATACATTGACCAAATTAGTAAGAAACTCTACAATTAAATCTTCATCATCATTACATTGA3540
ATTTAAGTAATTCTATAATAATATTCAAAGAATTCTCAAGATTATCATATTTCATAGAA3600
AAATAAACAAAGCATATGGATTAAGTTCTTGTTCATCAGTAAAATTATAATCTTCAGTA3660
GTAAATTATAAATTTCTCTCAAAGACATAGCATTTAAATCCTTACCAAAGTCATCGCAT3720
CTGCTTGTATATAATCACGATTTTTAGGATCACATAACAAATCATCATCACGAAATTCT3780
GCACATCACATATAGCACTCACTGGAGACACATAATATTTTCTTAATAACGCTTTTGTC3840
TTTTCCGTATGTATGCGAATTTCCCTGCCGAATAGGCTTTCTTTTCATAAAGTCTTCCG3900
TAGAACTGCCAGCATCTGATCTACGGCTAATTTTGTGCTCTTGCTGTTCACACTCATAG3960
AATCCGTGCAATCGGAGCTTGATACCATTTCTCTTTCTTTAAACTCTCTGGTCCATCGT4020
CACATTCATCGTTACTCGGTATTTTCCCACTTTCTCCTCTAACGTATATTGCACGCTCT4080
CCCGTTTTCGTACAAAGAAATAGATGAAGACATCGTACCAGTCTGTTCGCTTGAATTCC4140
AGATGAATTTGACTGGTTTGCCAGTTTTCTGAACAGATCCGAAGGGCTTGACTTGACTA4200
TCCAGATGTCCCTGCAACTGCGATTGGTGTAAGAGCAATCGTGGATGACGTGGATGTGA4260
CTCCTTCTTCAGAAAATCCGAACAGTCCGTTTCGTCTACTTCTTCCGTACTCACGCAAG4320
CGTCCAAACATTGATCACGGAGCTGAATATCTCGTAAGATAATGACATCGCTGATATAT4380
CGCTGGAAGGTTTAATACTTTGCCCAGTAACGTATCCAAAGAATTCACTGCCCATTTTT4440
CCAGTTCTTCTGCCATATATTGAAAGTTTTCTTGAGATTCTTTTGCAATGCTGGCCATT4500
TTCCTCGTCCTCCATGTGACCATTCACGTTTTCTAGATGTATTTGCCACCATGCTACAT4560
GTTCACTAGGGGATGTTCTTCCTGAAGACTCTCGTATAATGTCTTGATTAGGTCTGGTA4620
TACTATCGGTTTCTCTGTTGGTGTCTCCATTGTTCATCTGTAATTAATGTCTACTATTA4680
GATGTTTTACATAAAAACTATTAGACATATATTCGTCCTCTTCTGAACTACTTGAGTAT4740
TCCTTTTTTTATTAGGAGAGTTTTCATAAATTACAGATATATTAAACAATGGACAAGTA4800
GACATATTTGACACCAACTACTACCATCTTGTAATCTATTTATAATATCTTCAGCATCT4860
CTACGAACACAGTTCTATTATACATAAATCTGTATTCATCTGGATCTTCACAATTAGTA4920
AACAAAATTTACAAATTTTCCACATAGTTTTAAAGGGCTTTCCATTCATATTCCATGCA4980
TAAATTCATCAGTATCATAAGGGTCTCTATGGTTAATTAATTCTTTAAAATAATACACA5040
ATTCTATCAGCATAGTTTCATCTAACCATTCAGGTATTTCATTTAAATGCATAACAGCT5100
AATATAACTGGAAAGGTAATCTGTTTTGTTTTGTCCAATCCCAGTGTTCTAATTCTTCC5160
TAGCTAAAGTATGATCTATGTCTTCTCCACAAGCAATAATCTGATTATCTAGATCATGC5220
GCATATTAAGTATAGGTTTAGGCAAAATTGACAAGTCTAGACCATTAAGTCTAGCAGTC5280
TATAAGCCTCATAGAACAAAGACTTTGGATTGTACACTTTTTCAAATAAACGAACGAAC5340
CAAAGAAACCTGGCAATAGACATACCGATTATATTCTGGAACCACTTTTGCACAACACT5400
CTTTTTCACTGAGATGTTCACTCGACGACTGCTGCTCGTAGACTGATGATGGCGCTCTG5460
TGTTATCTCTATTTATAGCCAATGGTCCGCTGGCCAGCCACATCCTGTTTTGCACACGG5520
CCAGCCTCGACGCGAGTTTGCCGGTCAAACGAGGTCACAATAACAAGATAATAAAATGC5580
CATAAATTATCTCAAGGTTTGTTTGCATGCTATCTCTTYTACTCATATTGCTATCTCGC5640
CTAACAGTTGCTATAGTGCTATCTCACTCGCACACTATTGCTGTCCTTCACTAACATCA5700
GTCCTGTGTTCCACCTTCAGCTCCAAGGTCTTCGGATCCTCTCCATCATCAGGTCAGTA5760
ACCATCAGCTCAGAAGGTCAAAGGTCAGTAGACCAGAAGGTCACAGAAGTAGGTCAAGG5820
CATATAGAAGGTCAAAGGTCAAGGTCACCGTGACGTCATCAAGTACTCTATACATGCTG5880
GTACTTGCTTATACTCTACGAGTATTGCCN5910
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